Complete cures of various tumors like leukemias, lymphomas and solid tumors by the use of chemotherapeutic agents are not frequently achieved because of heterogeneous sensitivity of tumor cells to each antitumor agent. Cancer chemotherapy also fails because of intrinsic resistance of the tumor to various drug therapies. In other cases, a tumor may become resistant to the antitumor agents used in a previous treatment. The therapeutic effects of these agents are then lost. An even graver problem is that recurrent and relapsed cancers are resistant not only to the anticancer drugs used in previous treatments, but also manifest resistance to other antitumor agents, unrelated to the drug used previously either by chemical structure or by mechanism of action. These phenomena are collectively referred to as multidrug resistance.
The emergence of drug resistance represents a major obstacle to the successful treatment of cancers which can be treated by chemotherapy. To circumvent the problem of clinical drug resistance, chemotherapy may utilize combinations or successive treatments with functionally and structurally diverse antineoplastic agents to minimize the development of drug resistance and maximize the response to therapy. Despite this approach, most cancer patients relapse or never respond because of the development of drug resistance and further responses to therapy are limited (Chabner et. al., Cancer 54:2599-2608 (1984)).
The origins of tumor cell drug resistance in cancers which initially respond to chemotherapy are not fully understood. According to one theory, the somatic mutation theory, tumor cells are characterized by genetic instability (Goldie et al., Cancer Res. 44:3643-3653 (1984)), with observed frequencies of mutation to drug resistance of one in 10.sup.5 to 10.sup.7 (Bellamy et at., Cancer Invest. 8:547-562 (1990)). Since a detectable tumor mass of 1 cm.sup.2 (approximately 1 g) contains at least 10.sup.9 cells, it is nearly certain that some of the cells are resistant at the time of diagnosis prior to chemotherapy (Id.).
Cancer cells may become refractory to chemotherapy by several mechanisms. One type of drug resistance, called multidrug resistance, arises in cancer cells exposed to anticancer drugs derived from natural products (i.e., antineoplastic agents isolated from plants, fungi or bacteria). It can develop in cancer cells exposed to a single natural antineoplastic drug. In multidrug resistant (MDR) cells, cross resistance is observed to natural product antineoplastic agents (Vinca alkaloids, anthracyclines, epipodophyllotoxins, colchicine, actinomycin D and antibiotics) (See I. Pastan and M. Gottesman, New England J. Med. 1388, 1389 Table 1 (May 28, 1987)), but not alkylating anticancer drugs, bleomycin, or antimetabolites. The MDR phenotype is characterized by: (1) decreased intracellular accumulation of natural product anticancer drugs, secondary to their enhanced efflux; (2) cross resistance to other structurally and functionally unrelated natural product antineoplastic drugs; and (3) overexpression of a high molecular weight (150-170 kilodalton) transmembrane protein, termed the P-glycoprotein (the multiple drug transporter) which acts as drug transport pump. P-glycoprotein is an ATPase which functions by pumping structurally diverse antitumor drugs from cells. (See R. Fine and B. Chabner, Multidrug Resistance, in Cancer Chemotherapy, 117-128 (H. Pinedo and B. Chabner eds. 1986)(reviewed in Fine et al., Multidrug Resistance, Cancer Chemotherapy and Biological Response Modifiers, Eds: Pinedo, HM, Longo, DL and Chabner, BA. Elsvier Scientific Publications, NY, NY (1988)); Moscow et al., J. National Cancer Institute, vol. 80, 14-20 (1988); and Ford et al., Pharmacol. Rev. 42:155-199 (1990)).
A number of studies have implicated the P-glycoprotein in the MDR phenotype. The presence of the P-glycoprotein generally correlates with resistance in MDR cell lines (Kartner et al., Science 221:1285-1288 (1983)). The degree of resistance of certain tumor cells has been documented to correlate with both elevated expression of the drug transporter and reduced accumulation of antitumor drugs. (See A. Fojo et al., Cancer Res. 45:3002-3007 (1985).) Tumor cells expressing elevated levels of the multiple drug transporter accumulate far less antitumor agents intracellularly than tumor cells having low levels of the P-glycoprotein. Further, drug sensitive cells stably transfected with the mdr1 gene overexpress the P-glycoprotein and exhibit the MDR phenotype (Schurr et al., Cancer Res 49:2729-2734 (1989); Hammond et al., Cancer Res. 49:3867-3871 (1989); Sugimoto et al., Cancer Res. 47:2720-2726 (1987)). The role of P-glycoprotein as an energy dependent efflux pump is supported by the findings that depletion of cellular ATP in MDR cells eliminates the reduced drug accumulation defect of MDR cells (Dano, Biochimica et Biophysica Acta 323:466-483 (1973)), that the purified P-glycoprotein has ATPase activity (Hamada et al., J. Biol. Chem. 263:1454-1458 (1988)), and that expression of human DNA coding for P-glycoprotein confers high activity drug stimulatable ATPase activity (Sarkadi, B. et al., J. Biol. Chem. 267:4854-4858, 1992). Using antibodies to the P-glycoprotein, membrane vesicles prepared from MDR cell lines have been shown to contain this protein which is absent from membrane vesicles of parental drug sensitive cell lines by Western blot methods. The P-glycoprotein found in membrane vesicles of MDR cells shows specific binding of radiolabeled anticancer drugs and photoactive drug analogs. Membrane vesicles prepared from drug sensitive cell lines do not show specific binding of these compounds (Cornwell et al., J. Biol. Chem. 261:7921-7928 (1986); Cornwell et al., Proc. Natl. Acad. Sci., USA 83:3847-3850 (1986); Naito et al., J. Biol. Chem. 263:1187-11891 (1989)). Nucleotide sequence analysis of the mdr1 gene indicates that it codes for a 1280 amino acid protein with 12 transmembrane regions and 2 nucleotide binding sites. The deduced amino acid sequence of the P-glycoprotein shows extensive homology with the bacterial membrane transport protein for hemolysin B (Gerlach et al., Nature 324:485-489 (1986); Gros et al., Cell 47:371-380 (1986); Chen et al., Cell 47:381-389 (1986)).
A role for the P-glycoprotein in clinical drug resistance is suggested by several studies which have utilized specific monoclonal antibodies to the P-glycoprotein or cDNA probes to measure mdr1 RNA levels in tumors. High levels of the P-glycoprotein have been detected in drug-refractory hematologic malignancies, ovarian cancers, neuroblastomas, and sarcomas. These cancers usually respond initially to chemotherapy, but become refractory to further treatment and are considered to have acquired resistance. Other tumors documented to initially be drug-sensitive but to then become drug resistant include pheochromocytoma, acute lymphocytic leukemia in adults, acute nonlymphocytic leukemia in adults, nodular poorly differentiated lymphoma and breast cancer. High levels of the P-glycoprotein have also been found in untreated colon, renal, adrenal, and hepatic carcinomas which do not respond well to chemotherapy (reviewed in Bellamy et al., Cancer Invest. 8:547-562 (1990)). Interestingly, P-glycoprotein RNA expression is high in normal kidney, adrenal, hepatic and colonic tissues (Fojo et al., Proc. Natl. Acad. Sci., USA, 84:265-269 (1987)). These tissues have major roles in detoxification and secretion of toxins, suggesting a possible protective physiological role for the P-glycoprotein in normal tissues. Adult tumors derived from these tissues usually do not respond to chemotherapy. These types of tumors are considered to have intrinsic resistance. Other tumors documented to express high levels of the multidrug transporter include pancreatic, carcinoid, and chronic myelogenous leukemia in blast crisis. Increased levels of mdr1 expression have been found in untreated tumors derived from the colon, liver, kidney, adrenal gland, and pancreas which are considered to be intrinsically resistant (Goldstein et al., J. Natl. Cancer Inst. 81:116-124 (1989)).
It is likely that several mechanisms other than reduced drug accumulation may play a role in drug resistance. These include differences in DNA repair capacities, increased detoxification of anticancer drugs, alterations of the drug targets, and alterations of subcellular distributions of anticancer drugs which decrease drug concentrations at their targets (Fine, "Multidrug Resistance, Cancer Chemotherapy and Biological Response Modifiers", Eds: Pinedo, HM, Longo, DL and Chabner, BA. Elsvier Scientific Publications, NY, NY (1988); Moscow et al., J. National Cancer Institute, vol. 80, 14-20 (1988); Ford et al., Pharmacol. Rev. 42:155-199 (1990); Endicott et al., Annual Rev. Biochem. 58:137-171 (1989)). However, the P-glycoprotein is considered to be the major determinant of the MDR phenotype.
There is substantial evidence to suggest that P-glycoprotein function can be modulated by phosphorylation of the P-glycoprotein by cellular kinases, notably protein kinase C (PKC). Many MDR phenotype inhibitors are also PKC inhibitors. A number of laboratories have reported increased PKC activity in MDR cell lines and an association between PKC stimulation and the MDR phenotype (Palayoor et al. Biochem & Biophys. Res. Comm. 148:718-721 (1987); Fine et al., Proc. Natl. Acad. Sci., USA 85:582-587 (1988); Posada et al., Cancer Res. 49:6634-6640 (1989); Ferguson et al., Cancer Res. 47:433-441 (1987); O'Connor et al., Leuk. Res. 9:885-895 (1985); O'Brian et al., FEBS Lett. 246: 78-82 (1989); and Posada et al., Cancer Commun. 1:285-292 (1989)). In the MCF7 cell line, activation of PKC by phorbol ester has been shown to reduce intracellular accumulation of vincristine and doxorubicin, to transiently induce the MDR phenotype in sensitive, wild type (MCF7 wt) cells and to increase the MDR phenotype in the MDR MCF7 (MCF7 Adr 10) cells (Fine et al., Proc. Natl. Acad. Sci., USA 85:582-587 (1988)). The P-glycoprotein has been shown to be phosphorylated by a number of cellular kinases, including PKC (Yu et al., Cancer Commun. 3:181-189 (1991); and Chambers et al., J. Biol. Chem. 265:7679-7686 (1990)). Interestingly, phorbol esters, and verapamil, a calcium channel blocker, inducers and an inhibitor of the MDR phenotype, respectively, increased phosphorylation of the P-glycoprotein on different serine residues of the protein (Hamada et at., Cancer Res. 47:2860-2865 (1987); and Chambers et al., J. Biol. Chem. 265:7679-7686, 1990). Also interestingly, a cell line that was transfected with copies of the mdr1 gene did not express an MDR phenotype equivalent to adriamycin selected cells, yet expressed equivalent amounts of P-glycoprotein. When these cells were transfected with the .alpha. isoenzyme of PKC, their level of the MDR phenotype increased substantially, and was associated with P-glycoprotein phosphorylation and decreased drug accumulation (Yu et at., Cancer Commun. 3:181-189 (1991)). Reversal of the MDR phenotype probably involves multiple mechanisms, including inhibition of anticancer drug binding to P-glycoprotein, inhibition of P-glycoprotein efflux, and changes in P-glycoprotein phosphorylation.
There are two major strategies to overcome the problem of the MDR phenotype. One approach is to create new antineoplastic drugs or develop analogues of antineoplastic drugs currently used which are cytotoxic to MDR cancer cells. Two examples of anticancer drugs to which some MDR cells show less cross-resistance are mitoxantrone (Novantrone; see U.S. Pat. No. 4,197,249), a new antineoplastic agent, and morpholino anthracycline analogues of adriamycin and daunomycin. (Coley et at., Cancer Chemother. Pharmacol. 24:284-290 (1989)). The second approach to overcome the problem of the MDR phenotype is to identify resistance modifiers which reduce the degree of resistance in MDR cell lines in vitro. Such modifiers would be agents that inhibit the active efflux of antitumor agents by the drug transporter and/or agents that potentiate the efficacy of chemotherapeutic agents. The pharmacology and biochemistry of these types of resistance modifiers are extensively reviewed in Ford et at., Pharmacol. Rev. 42:155-199 (1990) and briefly summarized below for the most promising major resistance modifiers. These compounds are being or have been tested in clinical trials. They include calcium channel blockers, calmodulin antagonists, cyclosporins, and hormonal analogs.
Verapamil, a calcium channel blocker, was the first described resistance modifier and is probably the most potent in vitro resistance modifier previously known. Numerous investigators have described increases in accumulation and decreases in resistance to natural product chemotherapeutic drugs in a number of different MDR cells treated with verapamil and other calcium channel blockers. The mechanism by which verapamil, other calcium channel blockers, and calmodulin antagonists are thought to increase drug accumulation is by competing with anticancer drugs for binding to the P-glycoprotein, thereby inhibiting efflux of drug (Cornwell et al., J. Biol. Chem. 261:7921-7928 (1986); Cornwell et at., J. Biol. Chem. 262:2166-2170 (1986); Safa et at., J. Biol. Chem. 7884-7888 (1987); and Akiyama et at., Mol. Pharmacol. 33:144-147 (1988)).
Verapamil has been tested in Phase I and Phase II clinical trials (Benson et al., Cancer Treat Rep. 69:795-799 (1985); Preasant et al., Am. J. Clin. Oncol. 9:355-357 (1986); and Ozols et at., J. Clin. Oncol. 5:641-647 (1987)). However, the major problem associated with using verapamil to reverse drug resistance in patients is that it has dose-limiting cardiac toxicity due to blocking of the atrioventricular node. This toxicity prevents its use at concentrations required to reverse drug resistance in vitro. Thus, the lack of response observed in those studies may stem from the inability to achieve high enough concentrations to modulate clinical drug resistance. In a recent study, 3 of 6 patients with clinically resistant, P-glycoprotein positive myeloma responded to a regimen consisting of continuous infusion vincristine, adriamycin (i.e., doxorubicin) plus oral dexamethasone (VAD regimen) when verapamil was included in the regimen by continuous I.V. infusion. These patients had progressive disease while on the VAD regimen prior to the addition of verapamil (Dalton et at., J. Clin. Oncol. 7:415-424 (1989)).
Most studies of verapamil's effects on multidrug resistance have utilized racemic mixtures of the drug. The L form of verapamil is 10 times more active as a calcium antagonist than the D form of this compound, although both forms of verapamil increase drug accumulation to a similar extent (Gruber et al., Int. J. Cancer 41:224-229 (1988); and Mickish et at., Cancer Res. 50:3670-3674 (1990)). The use of the D form of this compound is an approach which may circumvent the obstacle of cardiovascular toxicity of calcium channel blockers, such as verapamil. However, more recent studies suggest that even D-verapamil is too toxic to use at high concentrations in humans.
A number of calmodulin antagonists have been found to be good resistance modifiers in vitro. Trifluoperazine, a phenothiazine antipsychotic drug, has been noted to increase drug accumulation and decrease resistance in MDR cell lines (Tsuruo et al., Cancer Res. 43:2905-2910 (1983); and Ford et al., Mol. Pharmacol 35:105-115 (1989)). Although the increases in drug accumulation were comparable to verapamil in these studies, verapamil was more effective in reversal of resistance to anticancer drugs in other studies (Tsuruo et at., Cancer Res. 43:2905-2910 (1983); and Ford et al., Mol. Pharmacol. 35:105-115 (1989)). In a clinical trial that combined oral trifluoperazine with constant infusion of adriamycin, 36 patients with tumors clinically resistant to adriamycin were treated. One complete response and six partial responses were noted. Neurotoxicity of trifluoperazine to the extrapyramidal tracts was dose-limiting. The plasma concentrations of trifluoperazine were approximately 10-fold less than the concentrations found to be optimal for modulation of chemoresistance in vitro (Miller et at., J. Clin. Oncol. 6:880-888 (1990)).
The thioxanthene class of antipsychotic drugs are similar in structure to the phenothiazines. The similarity is apparent when comparing the structures of thioxanthene and phenothiazine: ##STR1## A number of these compounds have recently been evaluated for the ability to reverse the MDR phenotype in MDR cell lines (Miller et at., J. Clin. Oncol. 6: 880-888, 1990.). Trans-flupenthixol, a thioxanthene, increased doxorubicin accumulation and reversed the MDR phenotype more effectively in a number of MDR cell lines than verapamil (Ford et at., Cancer Res. 50:1748-1756 (1990)). The trans isomer of flupenthixol was a much less potent antipsychotic in clinical trials than the cis isomer of this compound (Ford et at., Cancer Res 50:1748-1756 (1990)). It may be a more promising resistance modifier than trifluoperazine if it has less neurotoxicity.
The cyclosporins are immunosuppressive agents which can also potentiate toxicity of anticancer agents at clinically achievable concentrations. In some cell lines, it appears to enhance toxicity and increase drug accumulation in sensitive and resistant cells (Chambers et al., Cancer Res. 49:6275-6279 (1989)). Cyclosporin A (CsA) competitively inhibits vincristine and vinblastine binding to the P-glycoprotein (Tamai et at., J. Biol. Chem. 265:16509-16513 (1990)). Both Cylcosporin A and its nonimmunosuppressive analog, O-acetyl C.sub.9 -cyclosporin A (SDZ 33-243) inhibit [.sup.3 H] azidopine photoaffinity labeling of P-glycoprotein in intact MDR cells and in MDR membrane vesicles (Tamai et at.. J. Biol. Chem. 266:16796-16800 (1991)). The use of cyclosporin A to modulate drug resistance may be hampered by irreversible nephrotoxicity and immunosuppression in patients already compromised by myelosuppressive chemotherapy, whereas the use of nonimmunosuppresive cyclosporin analogs may be less toxic.
Hormonal analogs such as the antiestrogens tamoxifen and toremifene are employed in the chemotherapy of breast cancers. These compounds can also modulate resistance of estrogen receptor-negative MDR cell lines via estrogen receptor independent mechanisms (Ramu et at., Cancer Res. 44:4392-4395 (1984); and Bermin et al., Blood 77:818-825 (1991)). Tamoxifen and tamoxifen metabolites have been found to increase drug accumulation and decrease vinblastine resistance in intrinsically resistant renal cell carcinoma cell lines (Fine et al., Proceedings of the AACR Annual Meeting Abstract #2125 (1990)). The concentrations which elicited these responses were comparable to plasma concentrations achieved in patients participating in Phase I clinical trials to tamoxifen conducted at Duke University, which concentrations were associated with minimal neurologic toxicity that was reversible with cessation of tamoxifen and partial responses to the vinblastine-tamoxifen regimen (Trump, E. L. J. Natl. Cancer Inst. 84: 1811-1816, 1992).
In summary, a number of studies have demonstrated that multidrug resistance mediated by the function of the P-glycoprotein occurs in both cultured cell lines and human cancers. The identification of several pharmacologic agents which can antagonize the MDR phenotype in the laboratory has not, to date, identified resistance modifiers with good clinical efficacy, primarily due to doselimiting toxicity of the resistance modifiers. Thus there is a need in the art for means to overcome multidrug resistance expressed by tumors and for means to potentiate the effects of chemotherapeutic agents, in general.